Early detection of small tumors by non-invasive imaging holds the potential to substantially improve cancer patient survival and improve quality of life. However, early detection is currently limited to detecting masses composed of more than about one billion tumor cells. Improving upon this billion-cell threshold will require methods to deliver molecular imaging agents to tumors with increased selectivity, and to avoid the uptake of imaging agents in normal tissues. To reduce background and improve imaging contrast, a molecular imaging agent is proposed wherein tumor uptake will be amplified by the action of enzymes known to be selectively active in tumors. To accomplish this, we will first identify and affinity matures an antibody fragment with high affinity and specificity for a receptor present in breast tumors and other tumor types. The antibody will then be modified using protein engineering methods render binding activity dependent upon protease activity thereby generating protease- activated antibodies. In parallel, we will use protein display technologies to identify protease substrates that exhibit the requisite properties of activity, selectivity, and stability to enable precise in vivo targeting. Protease-activated antibody imaging probes will be designed to exhibit cross reactivity between human and mouse tumor receptor homologs to allow for the possibility of rapid translation to the clinic. The increase in selectivity and resultant increase in imaging contrast conferred by protease-activated antibodies will be assessed in mouse xenograft models by near-infrared imaging. This work is expected to yield highly-targeted, protease-activity imaging probes that exhibit increased selectivity for detecting multiple tumor types, and that could enable earlier tumor detection, or tumor phenotyping. More generally, this project will demonstrate a novel methodological approach that could be broadly applied to improve contrast in a variety of imaging applications. )